X-ray crystallographic studies reveal that the catalytic functions and substantial portions of the active site topologies of the vertebrate chymotrypsins and bacterial subtilisins are identical. These studies also indicate that the reactive conformation of covalent peptide inhibitors is the same for both classes of enzyme. We compared the reactivity of alpha- chymotrypsin towards the p-nitrophenyl esters of some semi-rigid 1,2,3,4- tetrahydro-2-naphthoic and indan-2-carboxylic acids alpha-substituents: NHAc; H; OAc) to that displayed toward the analogously alpha-substituted Beta- phenylpropionates. With the tetrahydro-2-naphthoates and Beta- phenylpropionates, the reactivity of alpha-chymotrypsin toward the substrates as a function of alpha-substituent and aromatic ring is quite similar. This may mean that the naphthoate esters are 'locked' into the same conformation assumed by specific substrates during their chymotryptic hydrolysis and suggests that the rigid compounds might prove useful as kinetic probes of the reactive conformations of natural substrates at the active sites of the subtilisins and other chymotrypsins. The reactivities of alpha- and gamma-chymotrypsins, and subtilisins BPN' and Carlsberg toward the rigid substrates and the analogous Beta- phenylpropionates will be measured under identical conditions. A complete set of binding and rate constant will be determined for each substrate with each enzyme using steady state and presteady state techniques. The data will be examined to determine whether the role played by the alpha-substituents and aromatic rings of the flexible substrates and their rigid analogs is the same with the subtilisins and chymotrypsins. The results of these experiments could lead to the detection of differences in substrate conformational requirements among the proteolytic enzymes surveyed. Conversely, they may establish that bound substrate topology is the same in the crystal and in dilute solution with all of these proteins, and that the reaction conformation of N-acylphenyl-alanine derived substrates is the same with the subtilisins and chymotrypsins. The latter would provide chemical support for the concept of convergent evolution of these proteases.